With the advent of whole genome sequencing, a new era of biology was ushered
in allowing for “systems-biology” approaches to characterizing microbial systems. The
field of systems biology aims to catalogue and understand all of the biological
components, their functions, and all of their interactions in a living system as well as
communities of living systems. Systems biology can be considered an attempt to
measure all of the components of a living system and then produce a data-driven model
of the system. This model can then be used to generate hypotheses about how the system
will respond to perturbations, which can be tested experimentally. The first step in the
process is the determination of a microbial genome. This process has, to a large extent,
been fully developed, with hundreds of microbial genome sequences completed and
hundreds more being characterized at a breathtaking pace. The developments of
technologies to use this information and to further probe the functional components of
microbes at a global level are currently being developed. The field of gene expression
analysis at the transcript level is one example; it is now possible to simultaneously
measure and compare the expression of thousands of mRNA products in a single
experiment. The natural extension of these experiments is to simultaneously measure and
compare the expression of all the proteins present in a microbial system. This is the field
of proteomics.
With the development of electrospray ionization, rapid tandem mass spectrometry
and database-searching algorithms, mass spectrometry (MS) has become the leader in the
attempts to decipher proteomes. This research effort is very young and many challenges
still exist. The goal of the work described here was to build a state-of-the-art robust MSbased
proteomics platform for the characterization of microbial proteomes from isolates
to communities. The research presented here describes the successes and challenges of
this objective. Proteome analyses of the metal-reducing bacteria Shewanella oneidensis
and the metabolically versatile bacteria Rhodopseudomonas palustris are given as
examples of the power of this technology to elucidate proteins important to different
metabolic states at a global level. The analysis of microbial proteomes from isolates is
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only the first step of the challenge. In nature, microbial species do not act alone but are
always found in mixtures with other species where their intricate interactions are critical
for survival. These studies conclude with some of the first efforts to develop
methodologies to measure proteomes of simple controlled mixtures of microbial species
and then present the first attempt at measuring the proteome of a natural microbial
community, a biofilm from an acid mine drainage system. This microbial system
illustrates life at the extreme of nature where life not only exists but flourishes in very
acidic conditions with high metal concentrations and high temperatures. The
technologies developed through these studies were applied to the first deep
characterization of a microbial community proteome, the deciphering of the expressed
proteome of the acid mine drainage biofilm.
The research presented here has led to development of a state-of-the-art robust
proteome pipeline, which can now be applied to the proteome analysis of any microbial
isolate for a sequenced species. The first steps have also been made toward developing
methodologies to characterize microbial proteomes in their natural environments. These
developments are key to integrating proteome technologies with genome and
transcriptome technologies for global characterizations of microbial species at the
systems level. This will lead to understanding of microbial physiology from a global
view where instead of analyzing one gene or protein at a time, hundreds of genes/proteins
will be interrogated in microbial species as the adapt and survive in the environment.
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